1. Technical Field of the Invention
The present invention relates to transgenic higher plants and transgenic plant cells thereof wherein the transgenic plant cells have been transformed with a gene encoding the translationally controlled tumor protein (TCTP). The transgenic higher plants grow about 30% faster than the parental plants during the juvenile growth stage.
The present invention relates to a method to engineer higher plants that can be more readily infected with Agrobacterial cells and from which more calli are induced in tissue cultures than the parental plants.
2. Description of the Prior Art
Plant growth and development are regulated by complex interactions between various environmental factors and endogenous developmental programs, such as plant growth hormones. Since plants are sessile, they have developed sophisticated systems to adapt themselves and to optimize their growth and development in response to ambient environmental conditions. Light is one of the most important environmental factors in that it is not only the sole energy source for plant growth but also regulates a variety of plant photomorphogenic responses, from seed germination to floral development (Kendrick et al. 1994). Plants therefore possess specialized photoreceptor proteins to precisely perceive light signals in the forms of wavelength, intensity, direction, and duration. Several photoreceptors that fulfil distinct physiological roles have been characterized so far. These include the red and far-red light absorbing phytochromes (Botto et al. 1996; Chory et al. 1996), the blue light absorbing cryptochromes (Ahmad et al. 1998; Christie et al. 1998; Cashmore et al. 1997), and the UV light absorbing UVA/B photoreceptors (Christie et al. 1996). Among them, the phytochromes are the best characterized. Phytochromes are molecular light switches that interconvert between two spectrally different forms, a photosensory red light absorbing Pr and a photoregulatory far-red light absorbing Pfr forms (Braslavsky et al. 1997; Song et al. 1996; Terry et al. 1995). Light signals perceived by phytochromes are subsequently transmitted through a series of downstream signaling components, such as G-proteins, Ca2+/calmodulin, protein kinase/phosphatase, cAMP/cGMP, and phytohormones and finally regulate genes involved in plant photomorphogenic responses (Neuhaus et al. 1997; Wu et al. 1997; Bowler et al. 1994a; Bowler et al. 1994b).
One primary role for the phytochrome photoreceptors is the regulation of plant growth and developmental process in earlier vegetative growth stage, such as stem and leaf growth, chlorophyll biosynthesis, and shade avoidance. However, plants in this growth stage are very vulnerable to environmental and pathogenic damages, mainly due to weak stems and leaves. This could result in a great economic loss, especially when agronomic plants are densely grown.
Although the molecular signaling pathway from light perception by the photoreceptors to physiological changes at cellular levels is largely unknown, many genes involved in this signaling pathway have been isolated and molecular biologically characterized. The translationally controlled tumor protein (TCTP) is one of the recently identified growth-related proteins in plants. The TCTP protein is a highly conserved cytosolic protein among various organisms, including man, animals, plants, and yeast (Woo et al. 1997). The TCTP proteins have been originally isolated from cancerous tissues in animals and from callus tissue and rapidly growing plant parts, such as apical stems and leaves, in plant, suggesting a regulatory role in cell proliferation (Woo et al. 1997; MacDonald et al. 1995; Hughes et al. 1993). However it has been later observed that it is also expressed in healthy animal tissues and that the expression is regulated by calcium ion at both the transcriptional and post-transcriptional levels (Wu et al. 1999; Sanchez et al. 1997). In accordance with this, it is notable that the TCTP has a Ca2+ binding activity (Sanchez et al. 1997). The TCTP protein is colocalized with the cytoskeletal microtubular networks (Gachet et al. 1999; Gachet et al. 1997) via association with xcex1- and xcex2-tubulins. It is interesting that the TCTP, which is otherwise a very acidic protein, has a basic domain of about 50 amino acids in the C-terminal region, which physically interacts with the tubulins (Gachet et al. 1999). Taken together, these observations suggest that the TCTP proteins have a housekeeping role in the regulation of cell growth and differentiation.
The TCTP genes have been isolated from several plants (Sage-Ono et al. 1998; Tamaoki et al. 1997; Woo et al. 1997). However, only the sequences of genes and gene fragments have been deposited in the databases without detailed molecular biological and functional analysis except for a few cases. The pea TCTP gene is actively expressed in rapidly dividing cells within root caps (Woo et al. 1997). In a short-day plant Japanese morning glory (Pharbitis nil cv. Violet), the TCTP MRNA accumulates to a high level when grown in the dark, but the expression level decreases to an undetectable level in the light (Sage-Ono et al. 1998).
To investigate the physiological role(s) of the TCTP in plant growth and development, we isolated a TCTP gene homolog from Nicotiana tabacum. The tobacco TCTP (referred to as ntTCTP in this work) protein physically interacts with the Pra3 small GTPase, a Rab-like GTPase originally isolated from Pisum sativum (Yoshida et al. 1993; Nagano et al. 1995). The ntTCTP-Pra3 interaction is GTP-dependent. The ntTCTP associates exclusively with the constitutively active GTP-bound Pra3, but not with the dominant negative GDP-bound Pra3. The ntTCTP gene is expressed in all tested plant organs, such as leaf, stem, root, and floral organs. Light does not exhibit any significant effects on the ntTCTP transcription, unlike that observed in the Japanese morning glory plant.
Transgenic higher plants of this invention grow much faster than the parental plants during the vegetative growth stage. They reach the adult stage in a shorter time (about 30% faster) and therefore potentially have less chance to be damaged by environmental factors compared to the parental plants. In addition, the transgenic plants are more rapidly regenerated and induce more calli from Agrobacterial infection in tissue cultures. Interestingly, transgenic plants with the pra3 small GTPase gene also showed essentially identical phenotypes as those with the ntTCTP gene, further supporting the specific Pra3-ntTCTP interaction.
With recent technical advances in plant tissue culture and gene manipulation, it is now a routine experimental technique to introduce a new gene into desired plants with an aim to improve productivity and quality. For example, vegetables can be engineered so that they grow faster or slower than the parental plants without affecting any other phenotypes. According to the present invention, the TCTP gene could be a good tool for the genetic manipulation of plant growth rate.
As used herein, the term xe2x80x9chigher plantsxe2x80x9d refers to multicellular differentiated organisms that are capable of photosynthesis. The term, therefore, does not include microorganisms, such as bacteria and fungi. The term xe2x80x9cplant cellxe2x80x9d includes any cell derived from a plant, including undifferentiated tissue, such as callus and plant seeds.
Accordingly, the object of the present invention is to provide a new tool to accelerate the growth rate of higher plants by transforming cells of higher plants with the TCTP gene. Such transgenic plants exhibit several desirable agronomic phenotypes. Since these transgenic plants have reduced transition time from seedlings to adult plants, they are less exposed to pathogens and environmental stress. This is a critical agronomic trait that significantly improves the productivity of economically important plants. The transgenic plants also have higher commercial value since they can be marketed earlier than the parental plants. While any agronomic plants can be subject to this manipulation to achieve embodiments of this invention, preferred plants could include cabbage, carrot, lettuce, spinach, onion, green onion, cucumber, tomato, potato, radish, tobacco, rice, cauliflower, melon, and watermelon. Woody plants also could be potential targets for this manipulation.
Provided also are transgenic higher plants that are more readily accessible to the Agrobacterium-mediated transformation. At least 2 times more calli are induced from the transgenic tobacco plants than from the parental plants under the identical experimental conditions. Furthermore the regeneration rate is much faster. These traits can be exploited in a way that cells of higher plants harboring the TCTP gene can be readily and efficiently transformed with transforming vectors containing other useful genes.
Also, provided includes an uninterrupted gene sequence encoding the ntTCTP protein, a nucleic acid fragment that can be directly ligated into recombinant DNA constructs, and ntTCTP expression vectors which can be used to transform cells of higher plants according to the invention.
This invention can be further utilized to improve the stability of recombinant proteins by fusing the TCTP polypeptide to a protein of interest. Many recombinant proteins, when overexpressed and purified from recombinant expression systems, are unstable and tend to be degraded, a troublesome difficulty frequently encountered with recombinant DNA technology. The recombinant ntTCTP protein expressed in E. coli is very stable against heat, salt, and pH and can be readily handled even at room temperature without any serious degradation and aggregation problems.